Conventional understanding of CD4 T cell development is that the MHC class II molecules on cortical thymic epithelial cells are necessary for selection, as shown in mouse models. Clinical data, however, demonstrate that hematopoietic stem cells reconstitute CD4 T cells in patients devoid of MHC class II. The difference ob-served in humans can be explained by our discovery that the CD4 compartment is efficiently reconstituted by MHC class II expressing thymocytes, demonstrating a novel thymocyte-driven pathway of CD4 T cell selection. We subsequently showed that, in mice, thymocyte-selected CD4 (T-CD4) T cells can produce both Th1 and Th2 cytokines immediately after stimulation, suggesting that they could be potent helper T cells. Unexpectedly, mice that have T-CD4 T cells are protected from airway inflammation as well as experimental allergic encephalomyelitis. In addition, our preliminary data showed that T-CD4 T cells suppress the generation of effector CD4 and memory CD8 T cells upon bacterial infections. Together, T-CD4 T cells seem to participate in various immune responses as a suppressor. Our long-term goal is to understand adaptive immune responses mediated by T-CD4 T cells. The objective in this application is to investigate how T-CD4 T cells suppress other target cells and how signaling through the thymocyte-thymocyte (T-T) interaction regulates the intrinsic function of T- CD4 T cells as a suppressor. The central hypothesis is that the T-T interaction via TCR-MHC class II and SLAM-SLAM delivers unique signaling, which changes the intracellular environments leading to the activation of a set of genes responsible for the suppressive function. The hypothesis, formulated based on our previous studies and preliminary data, will be tested by pursuing two specific aims: (1) Investigate the molecular mechanisms by which T-CD4 T cells function as a suppressor; and (2) Determine the role of TCR, SLAM/SAP, IL- 4, and MHC class II for the generation of CD4 T cells with the suppressor function. In Aim 1, in depth analyses of the T-CD4 T cell-mediated suppression function will be performed to characterize the T-CD4 T cell population and to compare it with that of Treg. In Aim 2, effects of signaling during the T-T interaction on the suppression function will be ascertained using BM transplantations followed by functional assays. The proposed research is innovative because it investigates an unexplored CD4 T cell population that bears a distinct and significant function. Undoubtedly, the proposed study is significant because it is expected to show the molecular mechanisms for the suppressor function of T-CD4 T cells. It is also expected to obtain a better understanding of the contribution of the T-T interaction to shaping the repertoire of T-CD4 T cells and the effector function of T-CD4 T cells. The outcome of the proposed research will provide new insights into how T-CD4 T cells modulate immunity and will facilitate the clinical translation of T-CD4 T cell mediated immunotherapy in the future.